Glucose and calcium contents and pH of WB or serum samples after centrifugation are shown in Fig. 1. Because glucose is contained in the ACD-A solution, glucose levels in the stored WB and serum samples (see Fig. 4c) after centrifugation were significantly greater than those of freshly collected WB samples. Total free calcium levels, including calcium chelated by citrate, in WB samples decreased significantly during storage and were significantly increased by addition of a 10% CaCl₂ solution. The pH levels of freshly collected WB samples were 6.0–6.5, and similar pH was observed in stored WB samples. Addition of the ACD-A solution (~10%) did not significantly decrease the pH of the stored WB samples. For reference, pH of ACD-A solution was 4.5–5.0.

The appearance of PRF clots prepared from freshly collected WB samples and WB samples stored for 2 days are shown in Fig. 2. There were no visual differences between these two PRF preparations. Microstructure of fibrin clots formed from fresh and 2-day-old WB samples is shown in Fig. 3. As for thickness and cross-links of fibrin fibers, no substantial differences were observed. For reference, fibrin clots that were prepared from PPP and bovine thrombin were composed of apparently thinner fibrin fibers as compared with PRF clots from either fresh or stored WB samples.

The biological activity was tested on human periosteal cells. The effects of PRF extracts on the cell proliferation are shown in Fig. 4a. PRF extracts (0–4%) prepared from fresh, 1-day-old, and 2-day-old WB samples exerted similar stimulatory effects on the proliferation of periosteal cells. PDGF-BB concentrations in PRF extracts prepared from fresh and stored WB samples are shown in Fig. 4b. PRF extracts and the supernatant serum fraction (see Fig. 4c) were subjected to measurement of PDGF-BB levels. The concentration of this representative growth factor of platelet concentrates [4] was significantly reduced in PRF extracts by storage. In contrast, PDGF-BB levels noticeably (but not significantly) increased in the supernatants.